Recently, reverse transcription polymerase chain reaction (RT-PCR) for dengue virus (DENV) has been reported to test positive in urine samples for a longer time frame than in serum. We evaluated two RNA extraction procedures from urine and investigated the stability of DENV RNA in urine and serum up to 1 year at different storage temperatures. In addition, 24 urine samples collected from patients with a recent infection were tested with DENV real-time RT-PCR and compared to the RT-PCR results on serum. Five patients with an acute DENV infection were followed up for 6 months by RT-PCR on urine. The automated extraction method with the MagNA Pure LC 2.0 device had a higher yield of DENV RNA compared to the manual QIAGEN method, explained by the higher volume used in the former method. DENV RNA in both serum and urine was stable at room temperature up to 1 month and at 4 °C and -20 °C for at least 1 year. The detection rate by RT-PCR on urine was 50 % (4/8) until day 7, 100 % (6/6) between 1 and 3 weeks after symptom onset, and 25 % (2/8) thereafter. Generally, DENV RNA concentrations are higher in serum than in urine up till day 7, switching to lower concentrations in serum thereafter. Peak concentrations in urine are reached around day 10, and RNA becomes undetectable 3 to 4 weeks following disease onset. This diagnostic tool is of added value in clinical settings by extending the period during which DENV infections are diagnosed by RT-PCR.